Vaccine for mystery swine disease and method for diagnosis thereof

ABSTRACT

The invention includes a vaccine and sera for treatment of Mystery Swine Disease (MSD), a method for producing the vaccine, methods for diagnosis of MSD, a viral agent that will mimic “mystery swine disease” and antibodies to the viral agent useful in diagnosis and treatment of MSD. The serum contains mammalian antibodies which are effective in treating MSD.

BACKGROUND OF THE INVENTION

[0001] Since 1987, the swine-producing industry has been subjected to adevastating epidemic of an unknown disease, often referred to as“Mystery Swine Disease” [MSD, more recently referred to as “SwineInfertility and Respiratory Syndrome (SIRS)], because researchers havebeen unable to identify the causative agent. MSD has affected hundredsof thousands of swine throughout North America and Europe. Once one pigis infected with MSD, that one pig can spread the MSD to an entire herdwithin three to seven days. From 1987 to 1991, the swine industry haslost millions of dollars in revenue as a result of MSD. A recent studyestimates that MSD causes a financial loss between $250 and 500 perinventoried sow.

[0002] MSD causes multiple symptoms in swine. The first symptom of MSDin a breeding herd of swine is usually anorexia and mild pyrexia. Inaddition, the herd animals may exhibit bluish discolorations in theirskin, especially in their ears, teats, snout, and the frontal portionsof their necks and shoulders. The affected skin may become irreparablydamaged. However, the most devastating symptom of MSD is thereproductive failure that occurs in a breeding herd of swine. MSD causessows to bear stillborn piglets; undersized, weak piglets withrespiratory distress; or piglets which die before they are weaned. Otherreproductive symptoms caused by MSD include early farrowing of piglets,a decrease in conception rates, failure in some sows to cycle, and areduction in the total number of piglets found in a litter. It has beenestimated that the number of pigs lost from reproductive failure isabout 10 to 15 percent of the annual production of pigs.

[0003] Research has been directed toward isolating the causative agentof MSD. A number of potential bacterial pathogens have been isolated.However, the types of potential bacterial pathogens have varied betweenswine-producing farms. Viral investigation has included fluorescentantibody examination, electron microscopic investigation, and serology.These methods have failed to locate the causative agent of MSD. As aresult, no one has yet developed a vaccine which can be used to treatMSD in the swine population.

[0004] Therefore, it is an objective of the invention to provide avaccine and sera which, when administered to a breeding swine herd, willreduce the presence of MSD in their population. Another object is toprovide a method of treating a population of swine with the vaccine toeradicate MSD from the swine population. Yet another object is toprovide a method for diagnosis of MSD.

SUMMARY OF THE INVENTION

[0005] These and other objects are achieved by the present inventionwhich is directed to a vaccine and sera for prevention and treatment ofmystery swine disease and to a method for its diagnosis in swine.

[0006] The vaccine is derived from an infectious agent that will infectswine with mystery swine disease (MSD). The infectious agent is obtainedfrom an inoculum of processed tissue of swine infected with the disease,preferably lung tissue. Preferably, the infectious agent is the productof an in vitro mammalian cell culture such as a simian cell lineinfected with the inoculum of the infected swine tissue. Preferably, theinoculum contains biological particles no greater than about 1.0 micronin size, more preferably 0.5 micron, most preferably no greater than 0.2micron. It is also preferable that the inoculum has been neutralizedwith antibodies to common swine diseases.

[0007] According to the present invention, a tissue homogenate obtainedfrom piglets in SIRS-affected herds consistently reproduced therespiratory and reproductive forms of SIRS when intranasally inoculatedin gnotobiotic piglets and pregnant sows. Gnotobiotic piglets soinoculated with-either unfiltered or filtered (0.45, 0.22, or 0.1 μm)inoculum became anorectic and developed microscopic lung lesions similarto lesions seen in SIRS-affected herds. The same inoculum also causedreproductive effects identical to those seen in SIRS-affected herds. Aviral agent has been recovered from the tissue homogenate. The viralagent causes a disease that mimics SIRS in piglets and pregnant sows.The viral agent has not yet been classified. However, the viral agent isa fastidious, non-hemagglutinating enveloped RNA virus. A viral agentcausing SIRS has been deposited on Jul. 18, 1991 with the American TypeCulture Collection in Rockville, Md. under the accession number ATCCVR-2332.

[0008] The serum for treatment of infected swine carries mammalianantibodies to the MSD. It is obtained from the blood plasma of a mammal(non-swine and swine) pre-treated with the above-described infectiousagent.

[0009] Alternatively, the serum is formulated from monoclonal antibodiesto MSD produced by hybridoma methods.

[0010] The method for diagnosis of MSD is based upon the use ofimmunospecific antibodies for MSD. The method calls for combination of afiltered homogenate of a lung biopsy sample or a biopsy sample orsimilar samples (homogenate or biopsy) from other tissue and theimmunospecific antibodies followed by application of a known detectiontechnique for the conjugate formed by this combination. Immobilizationor precipitation of the conjugate and application of such detectiontechniques as ELISA; RIA; Southern, Northern, Western Blots and the likewill diagnose MSD.

[0011] According to the present invention, therapeutic and diagnosticmethods employing antibodies to MSD involve monoclonal antibodies (e.g.,IgG or IgM) to the above-described fastidious, non-hemagglutinatingenveloped RNA virus. Exemplary antibodies include SDOW 12 and SDOW 17,deposited with the American Type Culture Collection on Mar. 27, 1992with accession numbers______and ______, respectively).

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 shows the cytopathic effects observed with SIRS virusVR-2332. FIG. 1A: Non-infected, unstained cell monolayer. FIG. 1B:Infected monolayer with small granular rounded and degenerating cellsobserved three days post-innoculation with the 6th passage of the SIRSvirus.

[0013]FIG. 2 shows the direct immunofluorescence staining of SIRSvirus-infected MA-104 cells. FIG. 2A: Non-infected cell monolayer. FIG.2B: Infected cells with intense, often granular cytoplasmic fluorescenceobserved three days post-innoculation.

[0014]FIG. 3 shows the density gradient profile of SIRS virus purifiedon CsCl density gradients. Peak virus infectivity occurs at 1.18-1.19g/ml. FIG. 4 shows an electron micrograph of virus particles observed inCsCl gradient fractions of density 1.18-1.19 g/ml. FIG. 4A: These fourparticles are spherical, 60-65 nm in diameter. Two particles are“empty”. showing electron-dense core (arrows), and the other twoparticles are complete. Bar =100 nm. FIG. 4B: Immuno-gold electronmicroscopy of SIRS virus with hyperimmune rabbit sera and anti-rabbitIgG labeled with gold particles. Note presence of core particleapproximately 25-30 nm in diameter within the virion. Bar=50 nm.

[0015]FIG. 5 shows the temperature stability of SIRS virus at 4° C.(open triangles), 37° C. (open circles), and 56° C. (closed circles).

DETAILED DESCRIPTION OF THE INVENTION

[0016] Determination of the cause of Mystery Swine Disease (MSD) hasbeen difficult. According to the present invention, however, theisolation and growth of the infectious agent causing MSD has beenachieved. As used herein, “infectious agent” refers to a virus capableof causing swine infertility and respiratory syndrome. Morespecifically, the infectious agent is a fastidious, non-hemagglutinatingenveloped RNA virus and zoopathogenic mutants thereof capable of causingswine infertility and respiratory disease in swine. The isolation of theinfectious agent is a major breakthrough and discovery. It enables theproduction of vaccines, antibody sera for treatment of infected swine,and diagnostic methods.

[0017] The vaccine is composed of an inactivated or attenuated MSDinfectious agent, derived from an inoculum processed from infected swinelung tissue or other swine tissue exhibiting the characteristic lesionsof MSD. Functional derivatives of infectious agent, including subunit,vector, recombinant, and synthetic peptide vaccines, or the like, arealso envisioned. A multi-step procedure is utilized in developing theMSD vaccine. The MSD infectious agent is first obtained as an inoculumby separation and isolation from infected swine tissue, preferably thelung tissue. The MSD infectious agent is then treated using knownvaccinological techniques to form a vaccine against MSD.

[0018] The MSD infectious agent is preferably isolated as an inoculatefrom lung tissue of pigs which exhibit rapid breathing due to the MSD(other tissue such as fetal tissue may also be used to recover virus).Such pigs are destroyed and their lung tissue removed. The lung tissueis then microscopically examined for thickened alveolar septae caused bythe presence of macrophages, degenerating cells, and debris in alveolarspaces. These characteristics indicate the presence of the MSDinfectious agent. Other swine tissue exhibiting lesions of this sort mayalso be used to isolate the MSD infectious agent.

[0019] The lung or other swine tissue is then homogenized with apharmaceutically acceptable aqueous solution (such as physiologicalsaline, Ringers solution, Hanks's Balanced Salt Solution, MinimumEssential Medium, and the like) such that the tissue comprises 10percent weight/volume amount of the homogenate. The homogenate is thenpassed through filters with pore diameters in the 0.05 to 10 micronrange, preferably through a series of 0.45, 0.2 and 0.1 micron filters,to produce a filtered homogenate containing the MSD infectious agent. Asa result, the filtered homogenate contains biological particles having asize no greater than about 1.0 micron, preferably no greater than about0.2 to 0.1 micron. The filtered homogenate can then be mixed withFreund's incomplete adjuvant so that the production of antibodies can bestimulated upon injection into a mammal. This mixture can be used as aninoculum for development of MSD in swine or further study of the MSDinfectious agent.

[0020] After obtaining a filtered homogenate containing the infectiousagent, the infectious agent can be inactivated or killed by treatment ofthe filtered homogenate with a standard chemical inactivating agent suchas an aldehyde reagent including formalin, acetaldehyde and the like;reactive acidic alcohols including cresol, phenol and the like; acidssuch as benzoic acid, benzene sulfonic acid and the like; lactones suchas beta propiolactone and caprolactone; and activated lactams,carbodiimides and carbonyl diheteroaromatic compounds such as carbonyldiimidazole. Irradiation such as with ultraviolet and gamma irradiationcan also be used to inactivate or kill the infectious agent.Alternatively, the infectious agent can be attenuated by its repeatedgrowth in cell culture from non-swine mammal or avian origin so that theability of the infectious agent to virulently reproduce is lost. Thedetails of the cell culture attenuation technique are given below.

[0021] The killed or attenuated infectious agent is then diluted to anappropriate titer by addition of a diluent adjuvant solution forstimulation of immune response. The titration is accomplished bymeasurement against MSD antibody in an immunologic test such as anELISA, RIA, IFA or enzyme substrate detection test as described below.

[0022] To produce a purified form of the infectious agent, the filteredhomogenate described above can be inoculated into a series of in vitrocell preparations. Cell preparations with mammalian organ cells such askidney, liver, heart and brain, lung, spleen, testicle, turbinate, whiteand red blood cells and lymph node, as well as insect and avian embryopreparations can be used. Culture media suitable for these cellpreparations include those supporting mammalian cell growth such asfetal calf serum and agar, blood infusion agar, brain-heart infusionglucose broth and agar and the like. Preferably the mammalian cells aremonkey kidney cells, most preferably African green monkey kidneyembryonic cells—monkey kidney cell line (MA-104).

[0023] After inoculating the cell preparation with the filteredhomogenate and growing the culture, individual clumps of cultured cellsare harvested and reintroduced into sterile culture medium with cells.The culture fluid from the final culture of the series provides thepurified form of the virulent infectious agent. Also, after a series ofrepeated harvests have been made, the culture can be grown, the culturefluid collected and the fluid used as an inoculum for a culture of adifferent cellular species.

[0024] In this fashion, the infective agent can be attenuated such thatthe culture fluid from the differing species culture provides thepurified form of the attenuated infectious agent.

[0025] Polyclonal antibody sera can be produced through use of theinfectious agent as an antigenic substance to raise an immune responsein mammals. The culture fluid or inoculum prepared as described abovecan be administered with a stimulating adjuvant to a non-swine mammalsuch as a horse, goat, mouse or rabbit. After repeated challenge,portions of blood serum can be removed and antigenically purified usingimmobilized antibodies to those disease specific antibodies typicallyfound in the serum of the bled animal. Further treatment of thesemi-purified serum by chromatography on, for example, a saccharide gelcolumn with physiological saline and collection of proteinaceouscomponents of molecular weight at least 10,000 provides a purifiedpolyclonal sera for use in treatment.

[0026] Monoclonal antibody sera can be produced by the hybridomatechnique. After immunization of a mouse, pig, rat, rabbit or otherappropriate species with MSD containing cell culture lysate orgradient-purified MSD as described above, the spleen of the animal canbe removed and converted into a whole cell preparation. Following themethod of Kohler and Milstein (Kohler et al., Nature, 256, 495-97(1975)), the immune cells from the spleen cell preparation can be fusedwith myeloma cells to produce hybridomas. Culturation of the hybridomasand testing the culture fluid against the fluid or inoculum carrying theinfectious agent allows isolation of the hybridoma culture producingmonoclonal antibodies to the MSD infectious agent. Introduction of thehybridoma into the peritoneum of the host species will produce aperitoneal growth of the hybridoma. Collection of the ascites fluidyields body fluid containing the monoclonal antibody to the infectiousagent. Also, cell culture supernatant from the hybridoma cell culturecan be used. Preferably the monoclonal antibody is produced by a murinederived hybrid cell line wherein the antibody is an IgG or IgM typeimmunoglobulin. Example monoclonal antibodies to the infectious agentfor SIRS are monoclonal antibody SDOW 12 and SDOW 17. In addition touses discussed elsewhere in this application, monoclonal antibodiesaccording to the present invention can be employed in various diagnosticand therapeutic compositions and methods, including passive immunizationand anti-idiotype vaccine preparation.

[0027] The vaccine of the present invention is capable of preventing andcuring MSD infections found in the swine population. For effectiveprophylactic and anti-infectious use in vivo, the MSD vaccine containskilled or attenuated MSD infectious agent and may be administered aloneor in combination with a pharmaceutical carrier that is compatible withswine. The vaccine may be delivered orally, parenterally, intranasallyor intravenously. Factors bearing on the vaccine dosage include, forexample, the age, weight, and level of maternal antibody of the infectedpig. The range of a given dose is 10³ to 10⁷ Tissue Culture InfectiveDose 50 per ml, preferably given in 1 ml to 5 ml doses. The vaccinedoses should be applied over about 14 to 28 days to ensure that the pighas developed an immunity to the MSD infection.

[0028] The MSD vaccine can be administered in a variety of differentdosage forms. An aqueous medium containing the killed or attenuated MSDinfectious agent may be desiccated and combined with pharmaceuticallyacceptable inert excipients and buffering agents such as lactose,starch, calcium carbonate, sodium citrate formed into tablets, capsulesand the like. These combinations may also be formed into a powder orsuspended in an aqueous solution such that these powders and/orsolutions can be added to animal feed or to the animals′ drinking water.These MSD vaccine powders or solutions can be suitably sweetened orflavored by various known agents to promote the uptake of the vaccineorally by the pig.

[0029] For purposes of parenteral administration, the killed orattenuated MSD infectious agent can be combined with pharmaceuticallyacceptable carrier(s) well known in the art such as saline solution,water, propylene glycol, etc. In this form, the vaccine can beparenterally, intranasally, and orally applied by well-known methodsknown in the art of veterinary medicine. The MSD vaccine can also beadministered intravenously by syringe. In this form, the MSD vaccine iscombined with pharmaceutically acceptable aqueous carrier(s) such as asaline solution. The parenteral and intravenous formulations of MSDvaccine may also include emulsifying and/or suspending agents as well,together with pharmaceutically acceptable diluent to control thedelivery and the dose amount of the MSD vaccine.

[0030] The method for diagnosis of MSD is carried out with thepolyclonal or monoclonal antibody sera described above. Either theantibody sera or the biopsied tissue homogenate may be immobilized bycontact with a polystyrene surface or with a surface of another polymerfor immobilizing protein. The other of the antibody sera and homogenateis then added, incubated and the non-immobilized material removed, forexample, by washing. A labeled species-specific antibody for theantibody sera is then added and the presence and quantity of labeldetermined. The label determination indicates the presence of MSD in thetissue assayed. Typical embodiments of this method include the enzymelinked immunosorbent assay (ELISA); radioimmunoassay (RIA);immunofluorescent assay (IFA); Northern, Southern, and Western Blotimmunoassay.

[0031] The following examples further illustrate specific embodiments ofthe invention. The examples, however, are not meant to limit the scopeof the invention which has been fully characterized in the foregoingdisclosure.

EXAMPLE 1

[0032] The MSD infectious agent may be characterized by determiningphysiochemical properties (size, sensitivity to lipid solvents, andsensitivity to protease) by treatment of the inoculum followed by theinoculation of gnotobiotic pigs to determine if the MSD infectious agentremains pathogenic.

[0033] A. Materials

[0034] Gnotobiotic pigs. Derivation and maintenance procedures forgnotobiotic pigs have been described in Benfield et al., Am. J. Vet.Res., 49, 330-36 (1988) and Collins et al., Am. J. Vet. Res., 50, 824-35(1989). Sows can be obtained from a herd free of reproduction problemsincluding MSD. Litters with stillborn and/or mummified fetuses shouldnot be used.

[0035] MSD inoculum (MN90-SD76-GP2, referred to herein as MNSD90×76-L orMNSD90×76-P). Trachea, lung, turbinates, tonsil, liver, brain, andspleen can be collected from nursing pigs in a Minnesota swine herdspontaneously infected with MSD (Collins et al., Minnesota SwineConference for Veterinarians, Abstract, 254-55 (1990)). A homogenate ofthese tissues (designated MN 89-35477) has been prepared in Hank'sBalanced Salt Solution without antibiotics and 0.5 ml can beintranasally inoculated into three-day-old gnotobiotic piglets using aglass Nebulizer (Ted Pella Co., Redding, Calif.). Inoculated piglets candevelop clinical signs and microscopic lesions similar to those observedin the spontaneously infected pigs. Lungs, liver, kidney, spleen, heartand brain from these gnotobiotic pigs can be collected eight days afterthe original inoculation and pooled to prepare another homogenate. Thissecond homogenate can then be inoculated one additional time ingnotobiotic pigs. Again, the same tissues may be collected andhomogenized, except that lung tissue can be prepared as a separatehomogenate because MSD can be ideally reproduced from the lunghomogenate. This lung homogenate represents the second serial passage ofthe original inoculum (MN 89-35477) in gnotobiotic pigs (Collins et al.,71st Meeting of the Conference of Research Workers in Animal Disease,Abstract No. 2 (1990)). Two filtrates can then be prepared using 0.20 μmfilter (Gelman Sciences, Ann Arbor, Mich.) and 0.10 μm filter (MilliporeCorp., Bedford, Mass.). These filtrates can be aliquoted and stored at70° C. All filtrates are free of bacteria and no viruses should beobserved on direct electron microscopy using negative stainedpreparations.

[0036] Control inoculum. Homogenates of lung tissues prepared from twomock-infected gnotobiotic pigs can be used as inoculum in control pigs.This control inoculum can be prepared as 0.20 and 0.10 μm filtrates asdescribed for the MSD inoculum.

[0037] Necropsy procedures and histopathology. Pigs can be euthanizedseven days after the original inoculation as previously described inCollins et al., 71st Meeting of the Conference of Research Workers inAnimal Disease, Abstract No. 2 (1990). Tissues can be collected, fixedin neutral buffered formalin, and processed for light microscopicexamination as described in Collins et al., Am. J. Vet. Res., 50, 827-35(1989). Specimens can be collected from turbinates, tonsil, trachea,brain, thymus, lung (apical, cardiac, diaphragmatic lobes), heart,kidney, spleen, liver, stomach, duodenum, jejunum, ileum, ascending anddescending colon, blood and mesenteric lymph nodes. These tissues can beprocessed and then examined using a light microscope to determinewhether lymphomononuclear encephalitis, interstitial pneumonia,lymphoplasmacytic rhinitis, lymphomononuclear myocarditis or portalhepatitis is present. Lesions can be consistently observed inspontaneously infected pigs from herds with MSD inoculum (Collins etal., Minnesota Swine Conference for Veterinarians, Abstract, 254-55(1990)). Fecal contents may also be collected and examined for virusparticles as previously described in Ritchie et al., Arch. Gesante.Virus-forsche, 23, 292-98 (1968). Blood can be collected for immunologicassays and tissues and cultured for bacteria as described in Example 3.

[0038] B. Infectious Agent Isolation

[0039] Lung tissue and combined brain-spleen-liver-kidney tissuesobtained from an infected piglet in an SIRS-infected herd werehomogenized separately. Ten percent homogenates of tissue were used. Theindividual homogenates were mixed with Minimum Essential Medium (MEM)containing gentamicin at about 100 μg per ml. Both samples werecentrifuged at about 4000×g for about 25 minutes. The supernatant wasthen removed and filtered through a 0.45 micron filter. The tissue andlung homogenates were then combined, and the combined material was usedto infect various tissue culture cell lines.

[0040] 1. In vitro testing. Two tests were conducted using 75 cm²plastic bottles. In test no. 1, the combined material was inoculatedinto two bottles of full cell sheet of each of the cell lines listedbelow. Additionally, to one bottle of each cell line about 2.5 mg oftrypsin was added. All other remaining conditions were the same for eachbottle of cell line. Serum was not in the culture medium. The inoculumwas 1 ml. All bottles were held for seven days at approximately 34° C.The results were recorded at the end of seven days. After freezing andthawing, a sample was taken for a second passage in the same cell line.The remaining material was frozen and stored at about −60° C.

[0041] In test no. 2, the combined material was inoculated into onebottle of the same cells as were used in test no. 1. However, the cellsheets were only 20-40 percent confluent at the time of inoculation. Themedia contained about 10 percent fetal calf serum. Again, the inoculumwas 1 ml, and the cultures were incubated at about 34° C. forapproximately seven days. The results of both test no. 1 and test no. 2are summarized below: Cell Line Used Test No. 1 Test No. 2 BovineTurbinate (BT) — — Feline Kidney (CRFK) — — Monkey (Vero) Kidney — —Monkey (Vero) Lung — — Canine Kidney (MDCX) — — Porcine (PK2a) Kidney —— Mink Lung — — Ferret Lung — — Bovine Lung — — Buffalo Lung — — BovineKidney (MDBK) — — Swine Testicle (ST) — — Monkey Kidney (MA-104) — +Human Rectal Tumor (HRT-18) — NT Human Lung NT —

[0042] There was no cytopathic effect observed in test no. 1 in any ofthe cell lines evaluated. In test no. 2, however, small clumps of MA-104cells began to swell and form “weak holes” in the monolayer around theedges of the bottle. Fluid was separated from the bottle, passed into anew bottle of MA-104 cells (again 20-40 percent cell sheet), and thensubsequently passed a third time. The cytopathic effect (CPE) becamestronger with each passage. The above-described procedures were repeatedfor the MA-104 cell line employing a full cell sheet. CPE was alsoobserved. Further testing demonstrated that the viral agent will alsogrow at 37° C. The presence of serum may be helpful for the initialisolation of the viral agent. Subsequent passages of the viral agent inthe MA-104 cell line will produce the CPE without the presence of serum.However, more pronounced CPE is observed with the use of serum in thegrowth medium for the MA-104 cell line.

[0043] The viral agent was passaged eight times in the MA-104 cell linewith good CPE developing in three days at passage five and greater. Thetiter obtained is approximately 5½ logs (10^(5.5)). The viral agent willalso grow in additional simian cell lines.

[0044] 2. In vivo testing. A third passage harvest was used to inoculatetwo three-day-old gnotobiotic piglets. Both piglets were exposedintranasally, one with 1 ml and the other with 2 ml. The piglets wereobserved for seven days, and then were euthanized.

[0045] Tissue samples were collected for histopathologic examinationsand for recovery of the viral agent. The histopathology report confirmedthat lung lesions in the infected piglets were identical to lung lesionsfrom piglets known to have SIRS. The tissue samples were processed asbefore, and then cultured on 20-40 percent and 100 percent monolayers ofthe MA-104 cell line with bovine fetal serum. The viral agent was againrecovered.

[0046] A third passage harvest was also used to inoculate sows in orderto verify that the reproductive effects of the disease can be duplicatedand confirmed. Two multiparous sows were inoculated intranasally at 93days of gestation. The sows delivered litters with 50 percent stillbirthpiglets (8/13 and 6/14 stillborn/live) on days 112 and 114 of gestation,respectively. Seven of the stillborn piglets were partial mummies andthe liveborn piglets were weak and failed to nurse vigorously. The viralagent was recovered from tissues of the stillborn piglets.

[0047] The viral agent has been recovered from three herds known to haveSIRS. Antibody titers to the ATCC VR-2332 agent have been identified inthese same herds.

[0048] Although there are some differences in clinical signs, i.e.,cutaneous cyanosis of the ears, tail and udder in European swine, theprevailing opinion is that the North American and European diseases arecaused by the same virus, a fastidious, non-hemagglutinating envelopedRNA virus as exemplified by the deposit ATCC VR-2332.

EXAMPLE 1A

[0049] Further Infections Agent Characterization

[0050] A. Materials and Methods

[0051] 1. Cells. Crandell feline kidney (CRFK), monkey kidney (MA-104)cells were grown at 37° C. in appropriate cell culture flasks. The CRFKand MA-104 cells were propagated in Eagle's minimum essential media(MEM) (available from Gibco Laboratories, Grand Island, N.Y.)supplemented with 10 percent gamma-irradiated fetal bovine serum (FBS)(available from JRH Biosciences, Lenexa, Kans.), 1 percentpenicillin-streptomycin and 2.5 μg/ml of amphotericin B. MA-104 cellswere propagated in the same media supplemented with 10 percent FBS and50 μg/ml of gentamicin. The FBS and cells were confirmed free of bovinevirus diarrhea virus (BVDV) using previously described methods of Mayeret al., Vet. Microbiol., 16, 303-314 (1988); Smithies et al., Proc.Annu. Meet. U.S. Animal Health Assoc., 73, 539-550 (1969); and Vickerset al., J. Vet. Diagn. Invest., 2,300-302 (1990).

[0052] 2. The source of the VR-2332 isolate (SIRS virus). The source andisolation of the SIRS virus for this Example is set forth below. Virusused in this study was on the 5th to 7th passage in MA-104 cells withtiters of 10⁵ to 10⁶ TCID₅₀/ml.

[0053] Gnotobiotic pigs. Gnotobiotic piglets obtained by closedhysterotomy were maintained in stainless steel tubs covered by flexiblefilm isolators as previously described by Miniatas O. P. et al., Can. J.Comp. Med., 42, 428-437 (1978). The isolators were maintained at anambient temperature of 30° C. and pigs were fed recommended amounts ofcommercial milk substitute three times a day. Fecal swabs were collectedprior to experimental inoculation and at necropsy, and were inoculatedonto sheep blood agar, tergitol-seven agar and brilliant green agar inaerobic and anaerobic atmospheres. Feces collected at necropsy were alsoexamined for viruses by negative contrast electron microscopy asdescribed by Richie et al., Arch. Gesante. Virus-forsche, supra.

[0054] Source of Inoculum. A 160-sow farrow-to-finish herd in WestCentral Minnesota experienced an outbreak of MSD with typical MSDsymptoms. A live sow, live neonatal piglets and stillborn fetuses weresubmitted to the Minnesota Veterinary Diagnostic Laboratory forexamination including gross necropsy, histopathology and routinemicrobial investigation. An inoculum was prepared for experimental usewith several tissues from clinically ill neonatal pigs. Morespecifically, two live and two dead 7- to 10-day-old piglets obtainedduring the epizootic from the affected herd were necropsied andspecimens were collected for diagnostic examinations. The live pigletswere euthanized by intravenous injection of euthanasia solution beforenecropsy. A 10 percent homogenate (MN89-35477) of brain, lung and tonsilpooled from each pig was prepared using Hank's Balanced Salt Solution(HBSS) containing 100 IU penicillin, 100 μg/ml streptomycin, and 5 μg/mlamphotericin B.

[0055] Experimental Transmission. A series of 14 gnotobiotic piglets waschallenged at three days of age with pooled tissue homogenates. Eachpiglet was challenged intranasally by use of a rubber bulb attached to aglass Nebulizer placed in front of the nares of the pig. Initially, twognotobiotic piglets were inoculated with 0.5 ml each of the unfilteredinoculum (MN89-35477), monitored for clinical signs of disease, and wereeuthanized by electrocution seven days post-exposure (PE).

[0056] A 10 percent homogenate (designated MNSD-1) of lung tissuespooled from the aforementioned gnotobiotic piglets was blind passaged byexposing each of three gnotobiotic piglets to 0.5 ml of homogenate, onepiglet receiving 0.5 ml of unfiltered homogenate, the second receiving0.45 μm filtrate, and the last one receiving a 0.22 μm filtrate. Thepiglets were euthanized by eight days PE and tissues were collected forhistologic examination, for further passaging in gnotobiotic piglets,and for virus isolation.

[0057] A 25 percent suspension of lung (MNSD90×76-L) and a composite ofbrain, liver and kidney (MNSD90×76-P) of the piglet inoculated with 0.45μm filtrate of MNSD-1 was prepared using phosphate buffered salinecontaining 0.5 mg/ml each of kanamycin, streptomycin, and vancomycin.Six gnotobiotic piglets were inoculated with lung homogenateMNSD90×76-L; four piglets received a 0.45 μm filtrate and two were givena 0.1 μm filtrate. Three uninfected, control gnotobiotic piglets wereinoculated, one piglet with a 0.45 μm filtrate of uninfected gnotobioticpiglet tissue homogenate in HBSS and two piglets with HBSS alone.

[0058] Virus Isolation. Tissue homogenates (MNSD90×76-L and MNSD90×76-P)were centrifuged at 1500×g at 4° C. for 20 minutes. The supernatant wasdiluted 1:1 with minimum essential medium (MEM) containing 10 μg/mlgentamicin, mixed thoroughly using a Vortex mixer and recentrifuged at4500×g at 4° C. for 30 minutes. The supernatant was collected andfiltered through a 0.45 μm filter. The filtrates from lung and tissuepool homogenates were combined and inoculated onto continuous cell lineMA-104. Virus isolation was done in 75 cm² flasks with 20-40 percentconfluent monolayers of MA-104 cells containing 50 ml of MEM (pH 7.5)with 10 percent fetal bovine serum (FBS). Cell cultures were maintainedat 34° C. for seven days. If no cytopathic effect (CPE) was observedwithin seven days, cultures were frozen, thawed and inoculated on MA-104cells and incubated as above.

[0059] Virus Titration. Virus titration was done in 96-well, flat-bottommicrotiter plates. Serial 10-fold dilutions of virus were prepared inMEM with 2 percent FBS. After three days, the cell growth medium wasdrained from the microtiter plates, 200 μl of the virus dilution wasplaced into each of five wells, and the plates were incubated at 37° C.in an atmosphere of 5 percent CO₂. After three days, media in wells withno CPE were replaced with MEM supplemented with 2 percent FBS (pH 7.5)and a final reading was made on the fifth day of incubation. Titers werecalculated by the method of Reed et al. in Am. J. Hyg., 27, 493-497(1938).

[0060] 3. Other viruses. Attenuated poliovirus, available from Dr. RogerKoment, Department of Microbiology, University of South Dakota School ofMedicine, Vermillion, S.Dak., was propagated on MA-104 cells to a titerof 10⁸TCID₅₀/ml and the Shope strain of pseudorabies virus, availablefrom National Veterinary Services Laboratory, Ames, IA, was grown onCRFK cells to a titer of 10⁵⁻⁷ TCID₅₀/ml. These viruses were used as RNAand DNA virus controls in studies to determine the nucleic acid type ofthe VR2322 isolate of SIRS virus.

[0061] 4. Preparation of antisera to VR-2332 isolate. Passage five ofthe VR2322 isolate of SIRS virus (titer 10⁶ TCID50/ml) was inactivatedwith 0.25 percent formalin, mixed 1:1 with Freund's incomplete adjuvant,and a rabbit was injected subcutaneously with 2 ml of this suspension attwo-week intervals for six weeks. Antisera prepared two weeks after thelast injection had a 1:512 neutralizing titer.

[0062] 5. Virus neutralization test (VNT). MA-104 cells were seeded inflat-bottom, 96-well microtiter plates for the VNT. Serial two-folddilutions of each serum (100 μl ) were prepared in MEM diluent and mixedwith an equal volume (100 μl ) of isolate VR-2332 containing 100-300TCID₅₀/100 μl . Each mixture was incubated at 37° C. for one hour, and200 μl of each serum-virus mixture was added to triplicate wells.Microtiter plates were incubated for an additional three days at 37° C.,examined by light microscopy for cytopathic effects (CPE), and theendpoint titer expressed as the reciprocal of the highest serum dilutionwhich neutralized CPE.

[0063] The following polyclonal antisera, unless indicated otherwise,were prepared as described for the VR-2332 isolate (except viruses werenot inactivated) and used in the VNT: the Miller strain of transmissiblegastroenteritis; porcine rotavirus serotype 4 (Gottfried); porcinerotavirus serotype 5-(OSU); porcine reovirus serotype 1; porcineenterovirus serotypes 1-8, available from National Veterinary ServicesLaboratory, Ames, Iowa; monoclonal antibody to porcine parvovirus,available from American Type Culture Collection, Rockville, Md.;encephalomyelocarditis virus, available from Dr. W. Christianson,Department of Clinical and Population Sciences, University of Minnesota,St. Paul, Minn.; pseudorabies virus, available from National VeterinaryServices Laboratory, Ames, Iowa; Eastern, Western and Venezuelan equineencephalitis viruses; monoclonal antibody D89 to BVDV described by Mayeret al., Vet. Microbiol., 16, 303-314 (1988); equine arteritis virus,available from National Veterinary Services Laboratory, Ames, Iowa;rubella; and lactic dehydrogenase virus, available from Dr. W. Cafruny,Department of Microbiology, University of South Dakota School ofMedicine, Vermillion, S.Dak.

[0064] 6. Direct electron microscopy (DEM). Cesium chloride gradientfractions were examined by DEM for virus particles as previouslydescribed by Benfield et al., J. Clin. Microbiol., 16, 186-190 (1982);Horzinek, “Non-arthropod-borne Togaviruses” (Acad. Press, London, 1981);and Richie et al., Arch. Gesante. Virus-i forsche, supra, and examinedon an electron microscope (Hitachi HU12A, Hitachi, Tokyo, Japan) at apotential of 75 kV.

[0065] 7. Immune electron microscopy (IEM). Immuno-gold labelling wasdone using goat anti-rabbit IgG gold colloidal particles (5 nm).Briefly, virus was concentrated at 40,000×g for 30 minutes at 4° C., thepellet was resuspended in 50 μl of distilled water, and 25 μl was placedon a piece of parafilm. A collodion carbon-coated grid was floated onthe drop of virus for 15 minutes, blotted dry with filter paper, andplaced on a 25 μl drop of rabbit anti-SIRS sera for two minutes. Gridswere washed in two changes of 0.1 percent bovine serum albumin(BSA)-Tris buffer for five minutes each and floated for 15 minutes on adrop of gold-labeled antirabbit-IgG. After six washes in BSA-Tris bufferfor two minutes each, and two washes in distilled water, the grids werenegatively stained and examined as described for DEM.

[0066] 8. Hemagglutination test (HAT). The ability of VR-2332 isolate tohemagglutinate sheep, goat, swine, cattle, mouse, rat, rabbit, guineapig, human type “O”, duck, and chicken erythrocytes was determined usingstandard methods. Two-fold dilutions of the 5th to 7th passage of theVR-2332 isolate of SIRS virus were prepared in phosphate-buffered saline(pH 7.2-7.4) in U-bottom microtiter plates. Equal volumes of a 1 percentsuspension of washed erythrocytes from each of the above species wereadded to each virus dilution, incubated at 4°, 22°, or 37° C.; and readafter one to two hours when erythrocyte controls (containing no virus)had settled into a button on the bottom of the well.

[0067] 9. Immunofluorescence (ImF). Indirect or direct ImF staining ofisolate VR-2332 infected and uninfected MA-104 cells was done using thepolyclonal or monoclonal antibodies tested by VNT. Swine influenza,available from National Veterinary Services Laboratory, Ames, Iowa (typeA, H1N1) and hog cholera virus polyclonal antisera, available. fromNational Veterinary Services Laboratory, Ames, Iowa, were also used.Scrapings of the cell monolayer were removed at 72 hours PI with asterile inoculating loop ImF staining as previously described byBenfield et al., J. Clin. Microbiol., supra. Positive control slideswere stained either with convalescent antisera from a sow (VNT titer1:256) or a monoclonal antibody (SDOW 12 or SDOW 17, described herein)to the VR-2332 isolate of SIRS virus.

[0068] 10. Filtration studies to estimate the size of isolate VR-2332.Clarified-infected cell culture supernatants were filtered through 0.45μm (Schleicher and Schnell, Keene, N.H.), 0.20 μm (Schleicher andSchnell, Keene, N.H.), 0.10 μm (Millipore Products Div., Bedford, Mass.)and 0.05 μm (Millipore Products Div., Bedford, Mass.) filters. Theinfectivity titer before and after filtration was determined using amicrotiter assay and a previously described method of Cottral, Manual ofStandardized Methods for Veterinary Microbiology, pp. 81-82 (CornellUniv. Press, Ithaca, N.Y., 1978). A 100-fold reduction in infectivitytiter was considered significant.

[0069] 11. Gradient purification of isolate VR-2332. The VR-2332 isolateof SIRS virus from clarified culture supernatant was concentrated bycentrifugation (Beckman SW 41 Ti rotor) at 200,000×g at 4° C. for 16hours through a discontinuous sucrose gradient consisting of 2 ml of 20,30, 40, 50, and 65 percent sucrose (wt/vol) in TNC buffer (10 mM Tris,100 mM NaCl and 2 mM CaCl, pH 7.8). Culture supernatants were alsoextracted with 1,1,2-trichlorotrifluoroethane (Sigma Chemical Co., St.Louis, Mo.), concentrated by ultracentrifugation at 100,000×g at 4° C.for one hour, and purified on cesium chloride density gradients (1.20g/ml) as described for the sucrose gradients. Fractions from eithergradient were harvested from the top, the refractive index determinedusing a refractometer, and selected fractions diluted in Hank's BalancedSalt Solution for virus titration as described above.

[0070] 12. Chloroform and fluorocarbon inactivation. Equal volumes ofSIRS virus (VR-2332) and chloroform were mixed periodically for 30minutes at ambient room temperature and then centrifuged at 500×g at 4°C. for 15 minutes. Similarly equal volumes of1,1,2-trichlorotrifluoroethane and SIRS virus were mixed on a vortex forfive minutes and centrifuged as described for the chloroform treatedvirus. After centrifugation, the aqueous phase of each treated samplewas harvested, diluted, and a microtiter assay used to determine theremaining virus infectivity. A 100-fold reduction in titer of treatedcompared to untreated virus was considered significant.

[0071] 13. Effects of inhibitors of DNA viruses on isolate VR-2332. Thecompounds 5-bromo-2-deoxyuridine (BUDR) and mitomycin C are knowninhibitors of the replication of DNA viruses, but do not inhibit RNAviruses with the exception of the Retroviridae (Easternbrook, Virology,19, 509-520 (1962); and Reich et al., Proc. Natl. Acad. Sci., 47,1212-1217 (1961)). Pseudorabies and poliovirus were used as the knownDNA and RNA virus controls in this experiment. Cells (CRFK or MA-104)were seeded in 96-well microtiter plates and duplicate wells wereinoculated with 100 μl of ten-fold dilutions of pseudorabies virus,poliovirus or SIRS virus, respectively. After a one-hour absorptionperiod at 37° C., media containing unabsorbed virus was removed andreplaced with either MEM (untreated virus controls); MEM supplementedwith 40 or 150 μg/ml of BUDR; or MEM containing 2, 10 or 20 μg/ml ofmitomycin C. Microtiter plates were incubated at 37° C. for anadditional four days, examined by light microscopy for CPE, and thevirus titer calculated as described previously by Cottral, supra (1978).A 1000-fold reduction in virus infectivity titer compared to theuntreated control was considered significant.

[0072] 14. Temperature stability of SIRS virus. A 2 ml aliquot of virusin MEM was incubated either at 4° C. or in water baths at 370 and 56° C.for at least five days. Aliquots from each tube at the three differenttemperatures were collected at 15, 30, 60 and 120 minutes and then at12-hour intervals from 12 to 120 hours. Virus samples were dilutedten-fold in MEM and virus titers calculated as described above.

[0073] B. Results

[0074] 1. Cytopathic effect of isolate VR-2332 on MA-104 cells. The CPEstarted as small, rounded clumps of cells, which appeared to be raisedabove the remainder of the uninfected monolayer (FIG. 1B). The number ofrounded cells increased and many cells became pyknotic and detached fromthe monolayer within two to four days PI. By five to six days PI, CPEwas evident in 100 percent of the monolayer. Infectivity titers variedfrom 10⁵ to 10⁷ TCID₅₀/1 ml on the 5th and 7th serial passage of virus,respectively. No CPE was observed in uninoculated MA-104 cells (FIG.1A).

[0075] Fluorescence was not observed in uninoculated MA-104 cells (FIG.2A). FIG. 2B demonstrates the intense, diffuse fluorescence observed inthe cytoplasm of inoculated MA-104 cells stained with eitherconvalescent sow sera, rabbit antisera or monoclonal antibody (preparedto the VR-2332 isolate of SIRS).

[0076] 2. Effects of lipid solvents on the infectivity of SIRS virus(isolate VR-2332). Pretreatment of virus with chloroform eliminatedinfectivity (titer reduced from 10⁵ to <10¹ TCID₅₀/1 ml), whereasfluorocarbon treatment had no significant effect.

[0077] 3. Hemagglutination. Virus did not hemagglutinate erythrocytesfrom 11 different species irrespective of the temperature of incubation.

[0078] 4. Estimation of the size of the SIRS virus by filtration. Virustiters were unchanged after filtration through 0.45, 0.20 and 0.10 μmfilters. However, infectivity titers were reduced 1000-fold (10⁵ to 10²TCID₅₀/1 ml) after passage through a 0.05 μm (50 nm) filter.

[0079] 5. Effects of DNA inhibitors on replication of the SIRS virus.Only the infectivity of the DNA virus, pseudorabies, was reduced byeither BUDR or mi tomycin C. The replication of poliovirus (RNA control)and isolate VR-2332 were not affected, indicating that the genome of theSIRS virus was probably RNA (Tables 1 and 2).

[0080] 6. Virus purification. Virus bands were not detected on sucrosegradients, and peak virus titers (10⁴ TCID₅₀/1 ml) were recovered fromfractions with buoyant densities of 1.18 to 1.23 g/ml. This peak virustiter was 1000-fold less than the infectivity titer (10⁷ TCID₅₀/ml) ofthe virus suspension before purification. Cesium chloride gradientsyielded a single, faint, opalescent band at a buoyant density,of 1.18 to1.19 g/ml. Peak virus infectivity of 1.3×10⁶ TCID₅₀/1 ml, which was 130times higher than peak infectivity from the sucrose gradient,corresponded to this visible band (FIG. 3).

[0081] 7. DEM of purified SIRS virus particles. Pleomorphic, butpredominantly spherical virions were observed by DEM in the CsClfractions (1.18 to 1.19 g/ml). Virions were 48 to 80 nm (average of 25particles=62 nm) in diameter and consisted of complete, electrontranslucent or empty (electron dense center) particles surrounded by athin membrane or envelope (FIG. 4A). [A 40 to 45 nm diameter isosahedralnucleocapid with short surface projections of about 5 nm in length wasobserved.] These particles contained cores that were 25 to 35 nm indiameter (FIG. 4B). Virions were immuno-gold labeled if the rabbithyperimmune antisera and gold-labeled anti-rabbit IgG were used asprimary and secondary antibodies (FIG. 4B). Virions were notgold-labeled in the absence of the rabbit hyperimmune antisera toisolate VR-2332.

[0082] 8. Antigenic relationship of SIRS virus to antisera from otherviruses. Antisera to known porcine viruses and various togaviruses didnot neutralize or react with SIRS virus antigen in infected MA-104cells. Convalescent sow and hyperimmune rabbit antisera had neutralizingantibody titers of 1:256 and 1:512, respectively.

[0083] 9. Effects of temperature on the infectivity of the SIRS virus.Virus infectivity for MA-104 cells was reduced 50 percent afterincubation for 12 hours at 37° C. and completely inactivated after 48hours of incubation at 37° C. and 45 minutes incubation at 56° C. (FIG.5). Infectivity was unchanged after one month incubation at 4° C. orfour months at 70° C. (data not shown). Lung tissue collected fromgnotobiotic pigs infected with SIRS virus and homogenized in Hank'sBalanced Salt Solution retains infectivity for pigs for at least 18months.

[0084] Results indicate that isolate VR-2332 is a fastidious,non-hemagglutinating enveloped RNA virus, which can be tentativelyclassified as a non-arthropod borne togavirus belonging to an unknowngenera.

[0085] The presence of an RNA genome of the SIRS virus was confirmed bythe ability of this virus to continue to replicate in the presence of5-bromo-2-deoxyuridine and mitomycin C, which are known to inhibit thereplication of DNA and one family of RNA viruses (Retroviridae)(Easterbrook, supra (1962); and Reich et al., supra (1961)), but notother RNA viruses. Our provisional classification of the SIRS virus asan RNA virus agrees with the observation that this virus replicates inthe cytoplasm of the cell as indicated by the presence of virus antigensdetected by ImF.

[0086] The VR-2332 isolate of SIRS virus is heat labile, but relativelystable for long periods of time at 4° and −70° C. The thermolability ofthis virus at 37° C. has practical applications for propagation of thevirus, suggesting that growth at temperatures lower than 37° C. willproduce higher virus yields. Refrigeration is sufficient forpreservation of diagnostic specimens for virus isolation for shortperiods of time, otherwise the sample can be frozen for several monthsor longer.

EXAMPLE 2

[0087] The purest form of an inoculum with the MSD infectious agent asdetermined from experiments in Example 1 may be used to transmit the MSDagent to pregnant sows to reproduce the reproductive form of the diseasesyndrome.

[0088] Discussion. Recently, transient anorexia and premature farrowing(both prominent clinical signs of MSD in the field) was induced in 2/2sows inoculated with the same MSD inoculum, which produces respiratorylesions in gnotobiotic pigs. In addition, 15/29 (52 percent) of the pigswere stillborns and the remaining 14 pigs were weak and did not nursewell. No gross or microscopic lesions were observed in the stillbornpigs or the placenta and isolation procedures to detect microbial agentsare now in progress. Therefore, the experiment described below testswhether the reproductive form of MSD can be transmitted to sows byintranasal inoculation and whether interference with fetal viabilityresults from replication in maternal tissues but not fetuses.

[0089] Experiments in Example 1 provide information on how the inoculumcan be treated (filter size, organic solvent extraction and/or proteasedigestion) to provide the purest form of the MSD agent for an inoculum(i.e., viral agent). Because the MSD agent has both a respiratory formin young pigs and a reproductive form in adult swine, it is necessary toreproduce the latter form of the syndrome to further verify that theinfectious agent is the putative cause of MSD.

[0090] A 93-day gestational sow is used as the experimental animalbecause it is possible to experimentally induce abortion in theseanimals inoculated with the MSD infectious agent. Sows can be purchasedfrom a commercial herd free of ongoing reproductive problems and MSD.Complete epidemiologic records on this herd can be computerized andinformation on gestation times, litter sizes, and average number ofstillbirths can be made available for comparative studies. Groups ofthree sows each can be intranasally inoculated at 93 days of gestationwith either the 0.20 μm filtrate (positive controls), a pathogenic butmodified inoculum as dictated by results from Example 1, a 0.20 μmfiltrate of the control inoculum (negative control), and a controlinoculum modified as indicated by results of experiments in Example 1.Each group of sows can be housed in separate isolation rooms andexamined daily until gestation is complete. Temperatures and clinicalsigns (anorexia, respiratory problems such as coughing, sneezing,panting, and increased respiration) can be noted daily. Sampling of sowscan be restricted to a pre- and post-farrowing blood sample forserology. The actual date of farrowing can be noted and the number ofstillborns, mummified fetuses, live “weak” pigs and live “normal” pigsdetermined. Fetuses can be examined for gross and microscopic lesions asdescribed in Example 1 and fetal tissues processed for microbiologicassays as described in Example 3. The fetal sera can also be assayed forthe presence of gammaglobulins and antibodies to PPV and EMCV (Joo etal., In Proceedings of the Mystery Swine Disease Committee Meeting,62-66 (1990); and Kim et al., J. Vet. Diagn. Invest., 1, 101-4 (1990)).Pigs born live can be observed for one week and morbidity and mortalityrecorded, after which these pigs can be euthanized and the tissuescollected for light microscopic and microbiologic examination asdescribed for the fetuses.

[0091] The 0.2 μm and the modified filtrates of the MSD inoculum arepathogenic for sows and induce anorexia, possibly a mild fever, andpremature farrowing with a large number of stillborn and weak pigs ineach litter. This illustrates evidence that the inoculum contains theMSD agent. Sows inoculated with the control inoculum farrow near termand have litter sizes within the normal range for the herd of origin asdetermined from the available epidemiologic database on this herd. Nolesions in the stillborn pigs are found and a high rate of mortalityamong the surviving weak pigs within one week after birth is observed.

EXAMPLE 3

[0092] Tissue samples collected from gnotobiotic piglets inoculated withthe MSD inoculum and euthanized at various times post-inoculation can beused to isolate and identify the MSD infectious agent to determine thesequential development of lesions, and to ascertain whether the MSDagent is immunosuppressive.

[0093] Immunofluorescence assays on frozen tissues and inoculated cellcultures. Immunofluorescence assays on frozen tissues and cell scrapingscan be done as previously described in Benfield et al., J. Clin.Microbiol., 16, 186-190 (1982) and Benfield et al., Am. J. Vet. Res.,49, 330-36 (1988). The frozen tissues and cell scrapings can be screenedfor PPV, EMCV (See Example 4 for definition of acronyms) and the MSDagent(s). Conjugates for PPV and EMCV are available at the South DakotaAnimal Disease Research and Diagnostic Laboratory. A hyperimmune sera ingnotobiotic pigs can be prepared from the purest form of the MSDinoculum. Two pigs can be inoculated intranasally, and then givensubcutaneous booster of the MSD inoculum in Freund's incomplete adjuvantat two and four weeks after the initial inoculation. Sera can beharvested from this pig two weeks after the last booster. A control serais also prepared in two gnotobiotic pigs using the control inoculum andthe same immunization protocol described for the MSD inoculum. Thesesera can be used as primary antibody and goat or rabbit anti-porcineimmunoglobulin conjugated with fluorescein isothiocyanate as secondaryantibody to detect MSD antigens in frozen tissue sections and cellcultures.

[0094] Serologic assays. Sera collected from control and inoculated pigscan be assayed for the presence or absence of antibody to PPV and SIV(hemagglutination inhibition), Leptospira (micro-agglutination), andEMCV (viral neutralization) (see Example 4 for definition of acronyms).Previous results indicated that serology to other common microbialagents were negative (Collins, et al., 71st Meeting of the Conference ofResearch Workers in Animal Disease, Abstract No. 2 (1990)).

[0095] Immunologic assays. Tissues and blood can be collected from MSDinoculated and control pigs so that their immunological status can bedetermined. Porcine leukocytes can be isolated from peripheral blood bysingle step discontinuous gradient floatation on Histopaque 1077 astaught by Pescovitz et al., J. Immunol., 134, 37-44 (1985). Cells fromlymph nodes, spleen and thymus can be spilled into single cells bymoderate mincing of the tissues and collection of the resultant cellsuspensions (Hurley et al., Cancer Res., 47, 3729-35 (1987)).

[0096] Porcine leukocyte phenotypes can be determined using a panel ofmonoclonal antibodies available through the American Type CultureCollection and Joan Lunney (USDA Beltsville, Md.) These includeantibodies for the measurement of total T cells, pCD2 (MSA4; Hammerberget al., Vet. Immunol. Immunopathol., 11, 107-21 (1986), helper/class IIMHC dependent T cells, pCD4 (74-12-4;Lunney et al., Vet. Immunol.Immunopathol., 17, 135-144 (1987), cytotoxic-suppressor/class I MHCdependent T cells, pCD8 (74-2-11; Ibid), macrophages and granulocytes(74-22-15A; Ibid), thymocytes and peripheral B cells, pCD1 (76-7-4;Ibid), and pig MHC class II antigens equivalent to human DRw (MSA3;Hammerberg et al., Vet. Immunol. Immunopathol., 11, 107-21 (1986)) andDQw (TH21A and others (VRMD, Pullman, Wash; Davis et al., HybridomaTechnology in Agriculture and Veterinary Research, Rowman and Allanheld,121-50 (1984)). Isotype-specific monoclonal antibodies to porcineimmunoglobulins are also available (Paul et al., J. Vet. Res ., 50,471-79 (1989)), and can be used at twice minimum saturatingconcentration for indirect fluorescent staining of leukocytes fromperipheral blood, lymph node, and Peyer's patches (Hurley et al., Vet.Immunol. Immunopathol., 25, 177-93 (1990)). To achieve two-coloranalysis, cells can also be stained with rPE-labeled avidin after beingtagged with biotin-bound (Pierce kit #21333) antibodies. Cells can beanalyzed by flow cytometry or a two-color analytical fluorescentmicroscope (PTI FSCAN system). Co-detection in the 488 nm laser line onthe flow cytometer or using the dual analytical fluorescent microscopecan easily be attained. Intensity of cellular fluorescence andpercentage of positive cells can also be determined.

[0097] In vitro functional assays such as lectin mitogenesis withconcanavalin A, pokeweed mitogen (PWM), and phytohaemagglutinin (PHA),can be performed as described in Hammerberg et al., Am. J. Vet. Res.,50, 868-74 (1989). Antigen-specific in vitro T cell responses tolysozyme can-also be modeled after their technique. B cell proliferativeassays can be performed with E. coli and S. typhimurium LPS oranti-immunoglobulin as reported in Symons et al., Int. Archs. AllergyAppl. Immun., 54, 10 67-77 (1977). In vitro antibody production, inducedwith PWM, can be accomplished and quantitated as described in Hammerberget al., Am. J. Vet. Res., 50, 868-74 (1989). Macrophage production ofIL-1 after 48-hour exposure to E. coli LPS can be measured in the mousethymocyte assay (Mizel, Immunological Rev., 63, 51-72 (1982)). IL-2production by PHA-stimulated lymphocytes can be measured as described byStott et al., Vet. Immunol. Immunopathol., 13, 31-38 (1986). Anisotype-specific anti-lysozyme ELISA can be done utilizing themonoclonal antibodies to porcine immunoglobulin isotypes (Paul et al.,Am. J. Vet. Res., 50, 471-79 (1989)).

[0098] To assess in vivo antibody production, three piglets inoculatedwith MSD and three inoculated with control inoculum can be injected witha 2 percent suspension of sheep erythrocytes and a 10 μg/ml solution ofbovine serum albumin at separate sites at 5, 7, 10, 14, and 24 daysafter their original inoculation. Pigs are euthanized at 24 days afterthe original inoculation, tissues are collected for histopathology asdescribed in Example 1, and blood is collected to assay for antibody?Thetotal antibody level and the specific IgG and IgM responses to eachantigen can be measured by antigen-specific radial immunodiffusion orELISA. Antigen-specific plaque assays can be performed on spleen cellsto assess the frequency of B cell clones in the infected and controlanimals (Kappler, J. Immunol., 112, 1271-85 (1974)).

EXAMPLE 4

[0099] Objective. The goal of this experiment is to prepare hyperimmuneantisera in a gnotobiotic pig to an isolate of MSD for use as adiagnostic reagent and for further characterization of the antigenicproperties of MSD.

[0100] Background. Previous studies done in gnotobiotic pigs at SouthDakota State University in collaboration with the University ofMinnesota indicated that pooled tissue homogenates from field case MN89-35477 induced lung lesions in gnotobiotic pigs. Pooled tissuehomogenates from these pigs have subsequently been used to produceclinical disease and respiratory lesions characteristic of MSD inthree-day-old gnotobiotic pigs (Collins et al., 71st Meeting of theConference of Research Workers in Animal Disease, Abstract No. 2 (1990)and Collins et al., Minnesota Swine Conference for Veterinarians,Abstract, 254-55 (1990)). Lung and tissue homogenates were prepared fromthis second passage of the original inocula in gnotobiotic pig (90×75)to produce a second inocula. The second inocula can be used as inoculaand antigen to produce the hyperimmune sera in this experiment.

[0101] Procedure to Accomplish the Objective

[0102] Gnotobiotic pigs. Gnotobiotic pigs can be derived and maintainedas previously described in Benfield et al., Am. J. Vet. Res., 49, 330-36(1988) and Collins et al., Am. J. Vet. Res., 50, 827-835 (1989).

[0103] Inoculation of hyperimmune sera. Hyperimmune sera can be preparedby initially inoculating one gnotobiotic pig as described above. Pigscan then be given a booster consisting of 1 ml of the second inocula and1 ml of Freund's Incomplete Adjuvant at 14 and 21 days after theoriginal inoculation (Harlow and Lane, 1988). The pig should then bekilled and exsanguinated 14 days or later after the last inoculation.Sera should be harvested and dispensed into appropriate aliquots andfrozen at −20° C.

[0104] Serology. The hyperimmune sera can be tested for the presence ofantibodies to common pathogens of swine as commonly done in mostdiagnostic laboratories. This sera can be tested for antibodies toHemophilus, Brucellosis, Leptospira (6 serovars), pseudorabies (PRV),parvovirus (PPV), encephalomyocarditis virus (EMC), and Swine InfluenzaVirus (SI).

[0105] Results. The pig used for preparation of the antisera was pig #4B(experimental number 90×238). This pig was inoculated on Nov. 1, 1990and observed daily for clinical signs until killed on Nov. 29, 1990.Clinical signs are summarized in Table 1. Unfortunately the continuingdegenerate condition of the pig mandated that it be euthanized afteronly one booster on Nov. 13, 1990. The pig was euthanized 16 days afterthe initial booster on Nov. 29, 1990.

[0106] Serology results were negative for all the above agents exceptPPV, which had a titer of 16, 384 (See Table 2). A pretitered sera onthis pig was not conducted.

[0107] Samples of lung, heart, brain, kidney, colon, small intestine,turbinates, spleen, stomach and trachea were collected when the pig wasnecropsied. These samples were evaluated and it was found that the lungsfrom this pig had lesions of severe pneumonia typical of that seen withfield cases of MSD.

[0108] The results of this experiment confirm initial studies that aninfectious agent is present in the second inocula because it can induceclinical disease and lesions typical of those observed in natural casesof MSD. TABLE 1 Date Observations of Inoculated Piglet (90 × 238) 10/29Surgery 11/1 Pig inoculated i.n. with 0.5 ml of above inocula usingNebulizer at 5:00 p.m. 11/2 Not observed. 11/3 This pig has 2 times asmuch milk in bowl as the control pigs. I'm not sure how well the pigsare eating or if the agent is the cause of the anorexia. Feces normal.11/4 May be a little slow but alert and strong, 1/2 milk left, fecessoft and brown. 11/5 9 a.m.: strong, alert, drank most of milk, fecesmucoid and brown (2). 4 p.m;: strong, alert, drank most of milk, fecesmucoid and brown (2). 11/6 9 a.m.: strong, alert, drank 1/2 of milk,feces brown mucoid (2). 6 p.m.: strong, alert, 1/2 of milk left, fecesloose yellow brown (2). 11/7 9 a.m.: strong, alert, drank most of milk,feces light brown mucoid (2). 3 p.m.: strong, alert, drank most of milk,feces light brown mucoid (2). 11/8 2 p.m.: Alert, does not drink milk aswell as controls, slower than controls, mucoid yellow feces. 11/9 8a.m.: Alert, still does not drink as aggressively as controls, pastyfeces. 5 p.m.: same observation as 8 a.m. 11/10 6 p.m.: alert, vigorous,rubs snout aggressively against feed pan, feces brown loose, not eatinglike controls. 11/11 6 p.m.: alert, vigorous, rubs snout vigorouslyagainst feed pan, feces pasty, not eating as well as controls, stillmilk in pan, by 6:30 p.m. controls had finished eating. 11/12 9 a.m.:alert, but not as aggressive as controls, after 5 minutes the controlshave cleaned pans but this pig still has at least 2/3 of milk in bowl.Some snout rubbing. 11/13 Inoculated with 90 × 75 lg & pool usingnebulizer (.5 m) and sp. IFA (1 ml) at 4 p.m. Alert but not asaggressive as controls, still has 1/2 pan of milk but controls haveconsumed all their milk. No snout rubbing, feces pasty. 11/15 7 p.m.:alert but not as aggressive as controls, still east slow, rough haircoat, but gaining weight like controls. 11/16 to Not much change, alertbut steady 11/24 declining in activity 1 p.m. (11/24): respiration seemsto be more rapid, hair coat is rough, not gaining weight like controls.11/29 Euthanized - blood collected for H.I. sera. Usual times collectedfor histopathology including tonsil. Blood collected for lymphocytemitogenic assays. No gross lesions noted. Set up turbinate explantcultures.

[0109] TABLE 2 Antibody Tests of Inoculated Piglet 1. Swine influenza -negative 2. Swine Encephalomyocarditis - negative 3. Swine APP -negative 4. Swine PRV - negative 5. Swine PPV - negative 6. SwineBrucella - negative 7. Swine Leptospirosa - negative 8. Swine EPI -negative

EXAMPLE 4A

[0110] Monoclonal Antibody Preparation

[0111] Mouse immunization. Eight weeks before the date of hybridomafusion, inoculate BALB/c AnN mice IP with a 1:1 suspension of antigen inFreund's complete adjuvant (CFA). The amount of antigen used isdependent on the immunogenicity and toxicity of the antigen. Use amaximum of 0.3 ml CFA per mouse. Five weeks after the initialimmunization, boost mice with antigen in CFA. One week prior to thefusion, immunize mice IP with antigen in saline. Two days before thefusion, inoculate mice IV with antigen in saline.

[0112] Myeloma cells. Maintain mouse myeloma cells (P3/NS-1/1-Ag4-1(ATCC TIB 18)) on Dulbecco's Modified Eagle Media (DMEM) with 10 percentfetal bovine serum. Approximately 10⁷ to 10⁸ cells are required forfusion with B cells obtained from one typical mouse spleen. Immediatelyprior to hybridoma fusion, harvest myeloma cells from a log phaseculture into a 50 ml centrifuge tube and pellet cells by centrifugationat 200×g for five minutes. Remove the supernatant and wash cells twicewith serum free DMEM followed by centrifugation as above. Resuspend thepellet (containing 10⁷ to 10⁸ cells) in 1 ml serum free DMEM.

[0113] Isolation of Spleen Lymphocytes

[0114] Euthanize the mouse by cervical dislocation and immerse in 70percent ethanol for several minutes. Remove the spleen by asepticprocedure and transfer it to a sterile petri dish containing 5 ml coldserum free DMEM. Use a scalpel blade to slit the spleen along the longaxis and gently scrape along the length of the spleen to release thesplenocytes into the media. Use a pipette to transfer the free cells andmedia to a 15 ml centrifuge tube, leaving the spleen casing behind.Allow the tissue debris in the tube to settle for five minutes andtransfer the single cell suspension to a fresh tube. Centrifuge cellsfor five minutes at 200×g, discard the supernatant and wash the pelletagain with cold serum free DMEM. Resuspend cells in 1 ml serum free DMEMand store cells on ice until fusion.

[0115] Fusion. Add the spleen cells to the centrifuge tube containingthe myeloma cells and centrifuge for five minutes at 500×g. Remove allthe supernatant and loosen the cell pellet by tapping on the side of thetube. Keeping all reagents and cells at 37° C., add 1 ml 50 percentpolyethylene glycol solution (PEG 4000, Gibco, Grand Island, N.Y.)dropwise to the tube over a one-minute period with gentle mixing. Allowthe mixture to stand at 37° C. for one minute. Add 1 ml warm serum freeDMEM dropwise over one minute with gentle mixing. Finally, add 20 mlserum-free DMEM dropwise over four minutes, then immediately centrifugecells at 200×g for five minutes. Discard the supernatant and resuspendcells in 47 ml DMEM containing 20 percent fetal bovine serum, 0.2units/ml insulin, 0.5 mM sodium pyruvate, 1 mM oxaloacetic acid, 2 mML-glutamine, non-essential amino acids, and 10 percent NCTC-109lymphocyte media. Add 1 ml volume of cell suspension to the wells of two24-well, flat-bottom tissue culture plates. Include a myeloma cellcontrol well and incubate plates at 37° C. and 10 percent CO₂ (SDMEM).

[0116] Following overnight incubation, remove 0.5 ml of media from eachwell without disturbing the cell layer. Add 1 ml SDMEM containing 1×10⁻⁴M hypoxanthine, 4×10⁻⁷ M aminopterin, and 1.6×10⁻⁵ M thymidine (HAT) toeach well and continue incubation at 37° C. and 10 percent CO². Continueto replace 1 ml of used media with 1 ml of fresh DMEM+HAT three timesweekly for two to three weeks. When significant clone growth isapparent, assay the wells for the presence of specific antibody byELISA, indirect FA, or other appropriate assay systems.

[0117] Cloning. Primary wells testing positive for specific antibodyshould be subcloned immediately to obtain a stable cell line and avoidovergrowth by other clones. Resuspend cells from selected primary wellsand perform cell counts using trypan blue stain and a hemocytometer.Make dilutions of the cells to obtain a final concentration of about 2cells/ml in SDMDM+HT. Use normal spleen cells obtained fromnon-inoculated mice as a feeder layer by adding 50 μl packed cells per100 ml media.

[0118] Add 250 μl of cell suspension to each well of 96-well plates andincubate at 37° C. and 10 percent CO₂. Clones should be visible in twoto three weeks and supernatants from wells containing single clones areassayed when significant growth is apparent. Repeat the cloningprocedure with wells testing positive for specific antibody. Slowlyexpand selected clones to tissue culture flasks for furthercharacterization and cryopreservation.

[0119] Ascites Production. Prime BALB/c AnN mice with 0.5 ml pristanegiven IP two weeks before inoculation with hybridoma cells. Harvesthybridoma cells and wash once with Hank's Balanced Salt Solution (HBSS).Resuspend cells in HBSS and inoculate primed mice IP with 10⁴ to 10⁶viable cells. When ascites production is apparent (usually one to twoweeks after inoculation) drain by inserting a 16 G 1-½″ needle ventrallyin the inguinal region. Hold the hub of the needle over a centrifugetube and drain ascites into the tube. Centrifuge ascites fluid at 200×g,filter through a 0.2 nm filter, and store frozen.

EXAMPLE 4B

[0120] Sirs Monoclonial Antibodies (SDOW 12 And SDOW 17)

[0121] The SDOW 12 (ATCC No.______ ) and SDOW 17 (ATCC No.______ )monoclonal antibodies to the SIRS virus were prepared using the abovestandard monoclonal antibody production protocol. The antigen used formouse immunization was passage 6 SIRS virus (VR-2332) grown on MA-104cells obtained from Boehringer Ingelheim Animal Health (BIAH). For eachimmunization, mice were inoculated with 0.3 ml of gradient purifiedvirus with a titer of 10⁶ TCID₅₀/100 μl .

[0122] An indirect fluorescent antibody (IFA) assay was used to detectspecific antibody in hybridoma primary wells and clone wells. Acetonefixed virus infected and non-infected cell monolayers in 96-well tissueculture plates were used for the IFA. Cell culture supernatants andascites fluids from these hybridomas produced bright, granularcytoplasmic fluorescence in SIRS infected cells.

[0123] Preliminary characterization of these monoclonal antibodiesincluded immunoglobulin isotyping and radioimmunoprecipitation of SIRSviral proteins. The SDOW 12 and SDOW 17 monoclonal antibodies are bothof the IgG₁ isotype. They also both bind to a 15 kD viral protein onradioimmunoprecipitation.

EXAMPLE 5

[0124] Three pilot studies are described. A gnotobiotic pig study wasundertaken to show that field material could be used to infect and causethe respiratory component of the syndrome in germ-free pigs. A secondstudy using conventional weaned pigs was undertaken to determine if therespiratory disease seen in gnotobiotic pigs could be reproduced inconventional pigs. And finally, a pregnant sow study was undertaken todetermine if the reproductive failure component of the syndrome could beexperimentally reproduced.

[0125] Material and Methods

[0126] Field case. See Source of Inoculum in Example 1A.

[0127] Gnotobiotic study. Six hysterectomy-derived gnotobiotic pigletswere inoculated intranasally at three days of age with the fieldinoculum (10 percent homogenates, various tissues). Filtered (0.22 μm)and unfiltered inoculum were used. Two control piglets were inoculatedwith media only. Clinical signs were monitored daily and the pigs wereeuthanized eight days post-inoculation, except one animal which was heldfor the production of hyperimmune serum. Tissues for virus isolation andhistologic examination were collected at necropsy along with sera whichwas screened for antibodies to leptospira, chlamydia, eperythrozoon,Aujeszky's disease virus, porcine parvovirus, encephalomyocarditisvirus, hemagglutinating encephalitis virus, swine influenza virus,bovine respiratory syncytial virus, canine distemper virus, bovine viraldiarrhea and hog cholera. The original inoculum and tissues fromgnotobiotic piglets were inoculated onto continuous and primary celllines for three passages. In addition, direct and immunoelectronmicroscopy was performed.

[0128] Conventional pig study. Three conventional 28-day-old weaned pigsfrom a farm with no history of MSD were intranasally inoculated with 10percent lung homogenates from affected gnotobiotic piglets. A lunghomogenate from a negative gnotobiotic pig was used as inoculum for acontrol. Piglets were monitored daily for clinical signs and werenecropsied eight days post-inoculation. Sera/tissues were processed asdescribed above.

[0129] Pregnant sow study. Eight multiparous sows with known historicaldue dates from a farm free of MSD were used in this study. Three weeksprior to the expected farrowing date, six sows were intranasallyinoculated with affected gnotobiotic lung homogenates and two sows withnegative lung homogenates. Clinical signs were monitored daily. The sowswere allowed to farrow naturally and when possible the farrowings wereattended in order to collect presuckled sera from live born pigs. Sowsand live pigs were euthanized shortly after farrowing and tissues werecollected for histopathology and virus isolation. Sera or fetal thoracicfluids were also collected.

[0130] Results

[0131] Field study. No gross lesions were seen at necropsy. Microscopicexamination of nursing piglets revealed necrotizing interstitialpneumonia and lymphomononuclear encephalitis. The fetuses did not havelesions but the sow did have a mild encephalitis. Microbiologicexamination did not yield conclusive results.

[0132] Gnotobiotic study. The piglets became anorexic and developedrough hair coats three days post-inoculation. Controls remained normal.Microscopic lesions were found in the principals inoculated withfiltered or non-filtered material. The lesions were similar to fieldcases and included: necrotizing interstitial pneumonia (6/6 [six out ofsix piglets]), lymphoplasmacytic rhinitis (4/6), lymphomononuclearencephalitis (2/6) and myocarditis (1/6). No etiologic agent wasidentified either by pre- and post-inoculation serology or throughinoculum/tissue examination.

[0133] Conventional weaned pig study. Clinically, the principals becamedull and anorexic two days post inoculation. The animals appearedchilled even though adequate heat was provided. One pig had an elevatedtemperature (41.5° C.) six days post-inoculation. Interstitialpneumonia, encephalitis and myocarditis were found in the principals butnot the control.

[0134] Pregnant sow study. Clinically, only two principals showed anysignificant temperature rises (1.5° C.) day three or fivepost-inoculation. However, anorexia was noted in 4/6 sows at day four orfive post-inoculation. Three sows farrowed up to seven days early andthree sows farrowed on time. Over 50 percent of the fetuses frominfected sows were born dead while the controls had normal litters. Bothstillborns and late-term mummies were found in infected litters.Laboratory findings were not conclusive—no specific agent has beenidentified and no lesions have been noted in fetuses to date.

[0135] Although no causative microorganism has been identified, thefindings suggest MSD can be transmitted experimentally to gnotobioticand conventional pigs (using field tissues from one farm). Both therespiratory and reproductive forms of MSD were reproduced. The agentinvolved appears infectious, filterable at 0.22 um and is seeminglyfastidious.

[0136] Discussion

[0137] MSD is an important emerging disease not only in the UnitedStates but also throughout the world. In order to study the disease in acontrolled setting, gnotobiotic pigs were inoculated intranasally atthree days of age with tissue homogenates from a farm experiencing theclinical signs of MSD. Microscopic lesions similar to field casesincluding necrotizing interstitial pneumonia and to a lesser extentlymphoplasmacytic rhinitis, lymphomononuclear encephalitis ormyocarditis were seen in principals but not controls. Using lunghomogenates from the gnotobiotic pigs, intranasal inoculation ofconventional four-week-old weaned pigs produced similar lesions.Multiparous pregnant sows were also inoculated with gnotobiotic lunghomogenates three weeks prior to their due dates. Clinically these sowswent through a period of anorexia and farrowed up to seven days early.Over 50 percent of the fetuses were either stillborn or in the beginningstages of mummification. The findings indicated the disease associatewith the infectious agent can be isolated and transmitted experimentallywith field tissues to gnotobiotic pigs and from gnotobiotic pigs toconventional weaned pigs or pregnant sows. This study provides a modelfor both the respiratory and reproductive forms of the disease whichwill lead to further investigations of the pathogenesis and diagnosis ofMSD.

What is claimed is:
 1. A vaccine suitable for use in prevention ofmystery swine disease, comprising: an inactivated or attenuatedinfectious agent derived from swine tissue infected with mystery swinedisease in combination with a pharmaceutical carrier.
 2. A vaccineaccording to claim 1 wherein the infectious agent is obtained from aninoculum of a filtered homogenate of swine lung tissue infected withmystery swine disease.
 3. A vaccine according to claim 2 wherein thehomogenate has been purified by neutralization with antibody sera toswine diseases selected from the group consisting of hemophilus,brucellosis, leptospire, parovirus, pseudorabies, encephalomyocarditis,enterovirus, swine influenza and any combination thereof.
 4. A vaccineaccording to claim 1 wherein the infectious agent is obtained from thecell culture medium of in vitro cultured, avian, insect, or mammalianorgan cells, primary or continuous, treated with an infectious agentisolated from the swine lung tissue known to have swine infertility andrespiratory syndrome disease.
 5. A vaccine according to claim 2 whereinthe filtered homogenate contains biological particles having a size ofno greater than 1.0 micron.
 6. A vaccine according to claim 5 whereinthe size is no greater than 0.5 micron.
 7. A vaccine according to claim1 wherein the infectious agent is a virus.
 8. A vaccine according toclaim 7 wherein said virus is a fastidious, non-hemagglutinatingenveloped RNA virus.
 9. A vaccine according to claim 1 wherein theinfectious agent is a biologically pure culture of swine infertility andrespiratory syndrome virus ATCC VR-2332 and zoopathogenic mutantsthereof.
 10. A vaccine according to claim 7 wherein the virus has beenpurified by gradient or serial cell culturation.
 11. A serum suitablefor treatment of swine infected with mystery swine disease, comprising:the semi-purified blood serum of a mammal inoculated with an infectiousagent derived from swine tissue infected with mystery swine disease. 12.A serum according to claim 11 wherein the infectious agent is obtainedfrom an inoculum of a filtered homogenate of swine lung tissue infectedwith mystery swine disease.
 13. A serum according to claim 11 whereinthe infectious agent is obtained from the cell culture medium of invitro cultured, avian, insect, or mammalian organ cells treated with aninoculum obtained from swine lung tissue infected with mystery swinedisease.
 14. A serum according to claim 13 wherein said culturedmammalian organ cells are a simian cell line.
 15. A method for diagnosisof mystery swine disease in swine, comprising: obtaining a lung tissuesample from the swine, forming a liquid homogenate of the sample, addingthe liquid homogenate to a vessel for immobilizing a viral material toform an immobilized mixture, adding to the immobilized mixture anonswine, mammalian species antibody serum to mystery swine disease toform a complex, adding a labeled anti-species antibody to detect thecomplex.
 16. A method according to claim 15 wherein the nonswinemammalian species antibody is a monoclonal antibody.
 17. A methodaccording to claim 15 wherein the labeled anti-species antibody carriesa radioactive or color producing enzyme label.
 18. A method according toclaim 16 wherein said monoclonal antibody is SDOW 12 or SDOW
 17. 19. Amethod for diagnosis of mystery swine disease in swine, comprising:forming a liquid homogenate of a sample suspected of harboring themystery swine disease infectious agent tissue sample from the swine,adding to the liquid homogenate an immunoreactive antibody which isimmunospecific for mystery swine disease virus, detecting the presenceof a precipitate of an antibody-antigen complex.
 20. A method accordingto claim 19 wherein said antibody is SDOW
 12. 21. A method according toclaim 19 wherein said antibody is SDOW
 17. 22. A method of making a MSDvaccine containing an inactivated or attenuated MSD infectious agent,the method comprising: identifying swine lung tissue having thickenedaveolar septae, degenerating cells and debris in aveolar spaces;homogenizing the swine lung tissue with a pharmaceutically acceptableaqueous solution to form a mixture; filtering the mixture through aseries of filters to produce a filtered homogenate containing the MSDinfectious agent; and inactivating or attenuating the MSD infectiousagent in the filtered homogenate to produce the MS vaccine.
 23. A methodaccording to claim 22, wherein the homogenate is filtered to containparticles having a size no greater than about 1.0 micron.
 24. A methodaccording to claim 22 wherein the size is no greater than about 0.5microns.
 25. A method according to claim 22 wherein the size is nogreater than about 0.1 microns.
 26. A biologically pure culture of afastidious, non-hemagglutinating, enveloped RNA virus, said culturebeing capable of affecting swine infertility and respiratory disease inswine, together with all zoopathogenic mutants thereof.
 27. A monoclonalantibody produced by a murine derived hybrid cell line wherein saidantibody is capable of specifically binding to at least one antigenicdeterminant of a fastidious non-hemagglutinating, enveloped RNA virus,capable of affecting swine infertility and respiratory disease in swine.28. A monoclonal antibody according to claim 27 wherein said antibody isan immunoglobulin molecule of the type IgG or IgM.
 29. A monoclonalantibody according to claim 27 designated SDOW
 12. 30. A monoclonalantibody according to claim 27 designated SDOW
 17. 31. A biologicallypure culture of swine infertility and respiratory syndrome virus, ATCCVR-2332, said culture being capable of affecting swine infertility andrespiratory disease in swine, together with all zoopathogenic mutantsthereof.
 32. A vaccine composition comprising modified, live swineinfertility and respiratory syndrome virus, ATCC VR-2332, and apharmaceutically acceptable carrier, which virus has been modified torender the virus non-zoopathogenic in swine.
 33. A vaccine compositioncomprising killed or inactivated swine infertility and respiratorysyndrome, ATCC VR-2332, and a pharmaceutically acceptable carrier.
 34. Amethod of immunizing swine against swine infertility and respiratorysyndrome which comprises administering to swine a vaccine composition asrecited in claim
 32. 35. A method of immunizing swine against swineinfertility and respiratory syndrome which comprises administering toswine a vaccine composition as recited in claim
 33. 36. A method ofgrowing and isolating swine infertility and respiratory syndrome virus,ATCC VR-2332, which comprises inoculating the virus on a full or partialsheet of simian cells in the presence of serum in a suitable growthmedium and incubating the inoculated cell sheet at about 34° C. to 37°C. until CPE is observed.
 37. The method as recited in claim 36 whereinthe simian cell line is MA-104.